Malaria is caused by protozoan parasites belonging to the genus Plasmodium. The life cycle of the parasite occurs in two phases--the asexual phase in vertebrates and the sexual phase in the mosquito (usually of the genus Anopheles). The four species of Plasmodium responsible for human malaria are P. falciparum, P. vivax, P. malariae and P. ovale.
Among these, the first two are the most common. P. falciparum causes the most severe form of malaria which in some instances is fatal. Further, this parasite also develops resistance to commonly used antimalarial drugs. The frequency of the cases caused by P. vivax varies between different countries but generally it is about 50-80%. The occurrence of P. malariae and P. ovale is rare.
The current method of diagnosis of malaria is by blood smear examination. This method is laborious and also requires expertise. Further, a skilled microscopist is allowed to examine a maximum of sixty slides a day. Diagnosis by serology may also be done but because of the persistence of antibodies current infections cannot be distinguished from past infections. The search for a new generation of diagnostic tests includes the possibility of detecting parasite nucleic acids as indicative of the presence of the parasite. Such a test requires very little blood (5-50 ul) that can be obtained from a finger prick, is sensitive and rapid. As few as 50 parasites in 10 ul of blood can be detected by nucleic acid hybridization (1). Hundreds of samples can be analyzed in a day with some initial training. The sensitivity of the assay enables the test to be used in blood banks for the screening of blood to be used for transfusion.
Nucleic acid hybridization could also be performed on insect tissue samples in order to identify the vector species as a carrier. Such information would help to intensify vector control measures in order to limit the geographic spread of malaria. Alternatively, chemoprophylaxis may be adopted in such areas and evaluation of this strategy may be accomplished using nucleic acid hybridization.
Double stranded DNA is of complementary nature. Under certain conditions of temperature and salt concentration, the complementary strands of DNA may be denatured or dissociated and reproducibly reassociated. The reassociation, may also occur between DNA and complementary RNA. The reassociating DNA and RNA may be tagged with detection devices (isotopic or non-isotopic) and appropriate detection methods may be employed. In the case of P. vivax, there is no report of any potential DNA probe sequences. The development of specific nucleic acid sequences to detect P. vivax would be important in order to determine the incidence of P. vivax in a geographically defined area. The term `probe` is used to denote a set of DNA sequences obtained biologically or synthetically.
Nucleic acid hybridization may be performed on any biological sample suspected of harbouring the parasite. Blood, for example, may be taken directly, solubilized in alkali and spotted on nitrocellulose or similar solid supports by standard methods (2). The DNA is immobilized on the supports and is brought in contact with the specific probe under appropriate conditions of temperature, ionic strength etc (3) which would favour specific reannealing of the probe and target nucleic acids. If the probe carries an isotopic tag (usually .sup.32 P), radioautography is performed to detect positive samples from negative ones. If the probe carries a non-isotopic tag e.g. biotin (4), an enzyme system e.g. avidin-alkaline phosphatase is employed and after a cascade of events colour develops in samples that are positive for the test.
In yet another technique (5), blood or the tissue sample to be analyzed may be collected directly in a chaotropic agent such as 4M guanidine thiocyanate. The hybridization process is performed in solution and the mixture is then filtered through a solid support under conditions which facilitate the binding of target-probe hybrids only while excess of probe does not bind to the matrix. Radioautography or colorimetric detection may be performed on this matrix. This hybridization format is especially useful when single stranded RNA probes are used.